CN1314363A - Purification of human myocardial troponin I and proparation of monoclonal anti-body thereof and its quick detecting method - Google Patents
Purification of human myocardial troponin I and proparation of monoclonal anti-body thereof and its quick detecting method Download PDFInfo
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Abstract
The present invention features that human myocardial troponin I is purified by means of affinity chromatography and is used to prepare its monoclonal antibody through mouse immunizing process, and that the one-step method for determining human myocrdial troponin I is established. The present invention has the advantages of good homology, high specificity and high sensitivity. The determining method is simple and low in cost and may be used widely in diagnosis of heart disease.
Description
The present invention relates to a kind of method and the monoclonal antibody method of this cardiac troponin I of preparation and the method that single stage method is measured its content of affine layer folding method purification of human cardiac troponin I.
Myocardial ischemic injury, particularly acute myocardial infarction (AMI) are one of principal diseases that threatens the human life.Striving to find good, the highly sensitive serum index of specificity clinically, so that it is diagnosed always.At present, creatine kinase has been used as the important indicator of diagnosing myocardial infarction with worker's acid (CK-MB), but it is peculiar that CK-MB is not a heart, also exist on a small quantity in normal people's the skeletal muscle, band muscle-derived disease is then more, non-cardiac surgery or Skeletal muscle injury patient often have CK-MB to increase, and easily cause AMI diagnosis false positive.Studies show that in recent years, the cardiac troponin I (Cardiac troponin I cTnI) has the myocardium specificity of height, when the myocardial cell is impaired, in the blood cTnI time of occurrence early, longer duration, closely related with myocardial damage degree and prognosis.It can be used as a kind of specific index of myocardial damage, and cardiopathic diagnosis is had crucial meaning.
Therefore, the human body cardiac muscle troponin I is carried out purifying and seeks its corresponding antibody, be used for heart patient's diagnosis has been become the front subject of current heart disease diagnosis research.Europe in 1999 and world's clinical chemistry association (IFCC) have proposed cardiac troponin I (cTnI) as the index of diagnosing AMI.
At present, the animal extraction is adopted in extraction to the cardiac troponin I mostly, still do not see the report that utilizes the human body cardiac muscle to extract, as Bodor GS (Bodor GS, Porter S, Landt Y, et al.The development of monoclonal antibodies and an assay for cardiactroponin I with preliminary results in suspected myocardial infarction.Chin Chem, 1992; 38:2203-2214.), Thulin E (Thulin E, Vogel HJ.Purification of rabbit skeletal muscle troponin C. Clin Chem Acta, 1988; 211-215.), Syska H (Syska H, Perry SV, Trayer. A new method ofpreparation of troponin I using affinity chromatography. FEBS Letters, 1974; 40; 253-257.), Pharmacia LKB biotechnology Ltd Co:(affinitychromatography principle and methods.1988, p15, Sweden.) document of Denging all has report to extracting the cardiac troponin I.But it utilizes animal to extract, and main drawback is that homology is poor, is used to cultivate the monoclonal antibody that produces the cardiac troponin I, and its specificity is relatively poor, and susceptibility is relatively poor.Be used for cardiopathic accuracy rate of diagnosis is still remained to be improved.
At present, prepare its antibody, prepare its monoclonal antibody or many anti-antibodys with animal cardiac muscle tissue extraction cardiac troponin I mostly with myocardium troponin I.(Cummins P such as Cummins, Young A, Auckland ML, et al. Comparison of serum cadiac specifictroponin I with creatine kinase, creatine kinase MB isoenzyme, tropomyosin, myoglobin and C reactive protein release in marathonrunners:cardiac or skeletal muscle trauma? Eur J Clin Invest, 1987; 17:317.) at first gave the injection of rabbit and sheep with myocardium troponin I in 1987 after, cultivate and prepared the polyclonal antibody of cardiac troponin I, and with the nucleic iodine labeling.Set up the quantitative measurment of radioimmunology to the cardiac troponin I.Adopt animal to prepare monoclonal antibody, be used for the content of human body cardiac troponin I, mainly exist the antibody homology poor, specificity, the relatively poor defective of susceptibility can't make the accuracy rate of heart disease diagnosis further improve.
At present, for the content detection of heart disease patient's cardiac troponin I, qualitative, semidefinite and quantitative three kinds of methods are arranged.Because the source difference of monoclonal antibody, reference point is not quite identical.Measuring principle is mainly based on various immunoenzyme detection methods and radioimmunity detection method.The external main immunoenzyme detection method of passing through utilizes the automated analysis instrument to finish (as automated analysis instrument such as Status, Access), can go out the result in 1 hour, but needs to buy the matched reagents such as the fine film of vinegar that scribble antibody, and such reagent costs an arm and a leg.Also have and adopt ELISA double fastener heart quantitative method to detect, but the generally system operation by hand of this method needs the long period, is unfavorable for the disease patient is striven for the valuable diagnoses and treatment time.
The purpose of this invention is to provide a kind of method of utilizing affinity chromatography purification of human cardiac troponin I, and utilize this cardiac troponin I to prepare its monoclonal antibody method.The method that provides cardiac troponin I that a kind of utilization obtains by this method and monoclonal antibody to set up single stage method rapid determination cardiac cardiac troponin I content simultaneously.
The objective of the invention is to realize by following technical solution:
(1) according to the present invention, the purification process of human body cardiac troponin I is as follows:
(1) gets the cardiac muscular tissue of the fresh or quick freezing and cold preserving of human body, degrease and fiber, after step process such as homogenate, centrifugal, precipitation, extracting, high salt dissolving, at chromatography media is CMSephadex-C50, Cellulose-DE52, buffer system is Tris-HCL, pH value is 7.2～7.8, and urea content is to carry out chromatography under the 6-8moL/L condition successively, extracts cardiac troponin C (cTnC);
(2) getting step (1) gained cardiac troponin C, is 0.1～0.3moL/LNaHCO at coupling solution
3, pH value is 7.8～8.5,3～6mmoL/L CaCL
2, level pad is 45～55mmoL/L Tris-HCL, 8～10moL/L urea, 0.8～1.2mmoL/LCaCL
2, 12～18mmoL/L β mercaptoethanol, pH value be under 7.8～8.2 the condition with the coupling mutually of Sepharose 4B affinity column, obtain Sepharose 4B affinity column;
(3) get slightly the carry product of above-mentioned steps (1) behind CM Sephadex-C50 chromatography, add step (2) gained Sepharose 4B affinity column again and carry out affinity chromatography, promptly get the purification product of cardiac troponin I.
(2) according to the present invention, the method for preparing monoclonal antibody of human body cardiac troponin I is as follows:
(1) gets aforementioned (one) prepared cardiac troponin I, repeatedly inject BALB/c mouse peritoneal immunity mouse, be 2～3 weeks pitch time, to the anti-cardiac troponin I of mice serum antibody titers greater than 1: 2000, get mouse boosting cell and SP2/0 myeloma cell;
(2) with two kinds of cell mixings of above-mentioned steps (1), add the DMEM serum-free medium and handle, add the HAT nutrient solution that contains 15～20% foetal calf serums again, put incubator and cultivate, carry out cytogamy;
(3) get the nutrient solution supernatant of above-mentioned steps (2), detect hybridoma in the nutrient solution, the i.e. specific antibody of cardiac troponin I with the ELISA method;
(4) get the detected hybridoma of above-mentioned steps (3) and adopt limiting dilution assay to cultivate, make the homology cell clone of propagation, i.e. monoclonal cell strain;
(5) the monoclonal cell strain that above-mentioned steps (4) is made contrasts with standard monoclonal antibody 2B1.9,2F6.6, adopts Western blot and ELISA method to measure, and detects anti-cardiac troponin I and catches monoclonal antibody IgG
1(being associated with the monoclonal antibody of horseradish peroxidase) and anti-cardiac troponin I mark biotin antibody IgG
2b(being associated with the monoclonal antibody of vitamin H), above-mentioned two monoclonal antibodies are the monoclonal antibody of the cardiac troponin I with high specific, hypersensitivity of present method gained.
(3) according to the present invention, the single stage method rapid assay methods of human body cardiac troponin I is as follows:
(1) with 50～150mmoL/L, pH value is that 9.4～9.6 bicarbonate buffer, streptavidin bag are by 96 hole polystyrene elisa plates;
(2) get testing sample and standard substance, with after the monoclonal antibody of the anti-cardiac muscle troponin I that is associated with vitamin H and the monoclonal antibody that is associated with the anti-cardiac troponin I of horseradish peroxidase contact, add above-mentioned elisa plate bag respectively respectively by in the hole;
(3) elisa plate of step (2) is hatched;
(4) get elisa plate after hatching, add substrate hydrogen peroxide and 2 at its bag respectively in by the hole, 2 '-Lian oxygen-two-3-ethylbenzene thiazole woods-6-sulfonate carries out enzymatic and develops the color;
Absorbancy when (5) reading above-mentioned steps (4) enzymatic colour developing back wavelength 405nm contrasts testing sample and standard substance, calculates the content of the cardiac troponin I in the testing sample.
Advantage of the present invention is: utilize the human body cardiac muscular tissue cardiac troponin I of purifying, gained cardiac troponin I is used for cultivating its corresponding monoclonal antibody of preparation, homology with gained antibody is good, the advantage that specificity and susceptibility are high, be used to detect the content accuracy height of cardiac's cardiac troponin I, can make cardiopathic accuracy rate of diagnosis is further improved.And can prepare in a large number with the monoclonal antibody that the present invention cultivates the cardiac troponin I, the success ratio height adapts to suitability for industrialized production.Adopting the present invention to prepare the monoclonal antibody of cardiac troponin I can substituting import one, reduces the expensive expense of import greatly, saves cost.Adopt the method for mensuration cardiac troponin I of the present invention in addition, operate very simply, and minute is fast, is generally to go out the result in 30 minutes, and is easy to utilize.Can be the Diagnostic Time of patient's saves valuable on the one hand, on the other hand, measuring method of the present invention need not to buy expensive automatic equipment, need not adopt expensive import reagent, greatly reduces the detection cost, has alleviated patient's cost burden.
Below in conjunction with embodiment aforesaid method of the present invention is described in further detail:
(1) the present invention is to the purification process of human body cardiac muscle troponin I:
1. the purifying of human body cardiac troponin C (cTnC)
(1) gets people's ventricle muscular tissue of-75 ℃ of storages behind the fresh or liquid nitrogen flash freezer of 100g, removal fat and fiber also shreds, homogenate buffer (the Tris-HCL 50mmoL/L.KCL 50mmoL/L that adds 3～5 times of volumes, EDTA 1mmoL/L, PH7.5) fully homogenate centrifugal 30 minutes, is removed supernatant, get precipitation, repeat above-mentioned steps 10 times; All need abundant homogenate at every turn.Get precipitation and use ethanol and ether extracting more than 2 times, drain into dry powder.Dry powder dissolved 12～24 hours with damping fluid 1moL/L KCL, and centrifugal 30 minutes of 10000g goes precipitation, gets supernatant;
(2) get above-mentioned supernatant 40-60% (NH
4)
2SO
4Precipitation.With the dissolving of 50mlCMSephadex C50 chromatography column level pad, and put in this damping fluid and dialyse 3 times, centrifugal 30 minutes of 10000g goes precipitation, gets supernatant;
(3) with the good CM Sephadex C50 dress post (2.5 * 30cm) of balance, 5 column volumes of level pad balance, with the clear sample (go up the sample protein content and be generally 200-400mg) of going up on above-mentioned (2), behind 5 column volumes of level pad balance, with NaCL 0-0.5moL/L gradient elution, automatic collector is collected, every pipe 6ml, speed 0.4ml/min;
(4) collect CM Sephdex C50 chromatography column NaCL gradient elution first protein peak, again through Cellulose-DE52 chromatography column NaCL0-0.3moL/L gradient elution, one albumen peak, back is cTnC after the balance liquid dialysis; 2. affinity chromatography purifying human cardiac troponin I
(1) preparation cTnC Sepharose 4B affinity column: get 50mg people cTnC (concentrating the back final volume is 20ml), put coupling solution (0.1moL/LNaHCO
3, pH8.3, CaCL
2, 5mmoL/L CaCL
2) the middle dialysis 3 times.Get the Sepharose 4B gel dry powder of 7.5g bromize hydrogen activating, in 15 minutes, wash and drain, with above-mentioned coupling solution washing and after draining, under 4 ℃, slowly shook 20 hours again with the 20mlcTnC mixing with 1moL/L HCL 1500ml.Be to drain after zero with aforementioned coupling solution washing to effluent liquid A280 value repeatedly after the taking-up.Be suspended in 0.1moL/L, in the Tris-HCL damping fluid of pH8.0, and under 4 ℃, slowly shook 12 hours.After taking-up is drained, use the 0.1moL/L acetate buffer (pH4.0 contains 0.5MNaCL) of 5 column volumes and 0.1moL/L Tris-HCL damping fluid (pH8.0 contains 0.5M NaCL) to wash 3 respectively and take turns.Use level pad (50mmoL/L Tris-HCL, 9moL/L urea, 1mmoL/L CaCL again
2, the 15mmoL/L beta-mercaptoethanol, pH8.0) washing, and (1.2 * 15cm) balances are standby to adorn post;
(2) affinity chromatography purifying people cTnI: get on the step 1 (1) clearly, put in the described coupling solution of step 2 (1) dialysis 3 times, last cTnC Sepharose 4B chromatography column.With the level pad flush away foreign protein of 50 times of column volumes, use elution buffer (50 mmoL/LTris-HCL, 9moL/L urea again, 10 mmoL/L EGTA, 15 mmoL/L beta-mercaptoethanol, pH8.0) wash-outs, automatic collector is collected, every pipe 2ml, speed 0.1ml/rain.The collection gained is the cardiac troponin I of purifying;
3.cTnI determination of activity and protein quantification: with two anti-ELISA sandwich method for determining cTnI activity, the Coomassie brilliant blue method is measured collection tube protein content, SDS-PAGE cataphoretic determination cTnI molecular weight.
(2) cardiac troponin I MONOCLONAL ANTIBODIES SPECIFIC FOR
1. immune animal: the BALB/c mouse of getting 8-12 age in week and myeloma cell system of the same race, to contain protein 100 μ g/ cTnI antigen and the abundant mixing of equivalent Fu Shi Freund's complete adjuvant only, in the injection mouse peritoneal, every 2 all 100 μ g/ cTnI antigen and abundant mixing of equivalent freund 's incomplete adjuvant only, repeatedly inject booster immunization in the mouse peritoneal.Above person can be used for merging at 1: 2000 for mice serum (indirect elisa method) after testing, titre, merged preceding 3 days booster immunizations once more in mouse peritoneal, dosage be 50 μ g/ only; 2. cytogamy:
(1) get the 40mlHAT nutrient solution, 15mlDMEM serum-free medium and 1ml50%PEG (M12000) place the pre-temperature of 37 ℃ of water-baths respectively;
(2) get BALB/c mouse SP2/0 myeloma cell (2-5 * 10 of above-mentioned immunity respectively
7), splenocyte (10
8) suspension adds mixing in the 50ml centrifuge tube, and add the DMEM serum-free medium to 40ml.Centrifugal 10 minutes, use up supernatant liquor, be mixed into pasty state;
(3) centrifuge tube is placed 37 ℃ of pre-warm beakers that are filled with water, get the 50%PEG solution of the pre-temperature of 0.7ml, add in 1 minute, left standstill for 90 seconds.Drip the serum-free medium of the pre-temperature of 37 ℃ of 15ml immediately, make the PEG dilution and fail.The dropping method is to add 1ml in preceding 30 seconds; The back added 3ml in 30 seconds; In 1 minute, add then;
(4) add the DMEM serum-free medium to 40ml, centrifugal 10 minutes, use up supernatant liquor.Add the HAT nutrient solution that 40ml contains the 15%-20% foetal calf serum.With suction pipe mixing gently, be added drop-wise in the aperture of 4 96 porocyte culture plates that contain feeder cell, 37 ℃, 7%CO are put in 2 in every hole
2Incubator in cultivate;
3. the selection of hybridoma is cultivated
Immune mouse spleen cell and murine myeloma cell after PEG handles, form the mixture of various kinds of cell composition, comprising the myeloma cell and the immune spleen cell that do not merge; Myeloma cell's the coenocyte and the coenocyte of immune spleen cell, and the heterokaryon of myeloma cell and immune spleen cell.Only the latter could form hybridoma.For this reason, in this various kinds of cell mixture, must remove the cell of not fusion and the coenocyte that allogenic cell merges, and select real hybrid cell.Therefore, changing liquid with aforesaid HAT nutrient solution in the 1st, 3,5,7 days after cytogamy cultivates;
4. the detection and the hybridoma cell cloneization of specific antibody
Draw the supernatant liquor of each culture hole, detect the culture hole of the specific antibody that contains cTnI in the nutrient solution with indirect elisa method.Adopt limiting dilution assay to make hybridoma cell cloneization.Can breed and be the homology cell clone through cultivating the back individual cells;
5. hybridoma is frozen
Hybridoma should be frozen as early as possible once setting up, preserve hybridoma not reason go down to posterity and pollute or lose because of variation.Must be after obtaining the hybridoma of secreting specificity antibody promptly preserve several cells in early days in liquid nitrogen.The myeloma cell strain that is used for cytogamy is also used with the method preservation, to obtain reliable and stable parental cell source.
It is vigorous to get growth, and the good cTnI cloning cell of form is made cell suspension, and centrifugal 5 minutes, remove supernatant liquor, add 4 ℃ frozen storing liquid (9 parts of complete culture solutions add 1 part of dimethyl sulfoxide (DMSO) DMSO), finally making cell density is 3-5 * 10
6Cell/ml is sub-packed in prolonged preservation in the frozen or liquid nitrogen of the frozen pipe of 2ml with the 1ml cell suspension;
6. a large amount of preparations of monoclonal antibody
Before the inoculation hybridoma, give BALB/c mouse abdominal injection 0.5ml pristane (pristane) or whiteruss earlier, then every mouse peritoneal injection 5 * 10
5-10
6Individual hybridoma.The inoculation hybridoma is got its ascites to 7-10 after day, and centrifugal 10 minutes, draw supernatant liquor, add 0.02% sodium azide, packing is stored in-70 ℃;
7. Purification of Monoclonal Antibodies
Monoclonal antibody is energy and albumin A combination under high pH environment, and can be from wash-out on the albumin A under low pH environment, and operation steps is as follows:
(1) gets 5ml Protein A-Sepharose (Repligen, Cat.No.IPA-300) strutting.50ml binding buffer liquid stream is washed;
(2) get 10-15ml ascites and equivalent PBS damping fluid (phosphoric acid salt 100mmoL/L, pH7.4) mixing strutting.Hybridoma supernatant transfers to strutting behind the pH9.0 with NaOH.50ml binding buffer liquid stream is washed;
(3) elution buffer wash-out, every pipe is collected the 0.1M tris of 1ml and equivalent, pH9.0 mixing;
(4) A280 colorimetric.Collect the peak pipe and merge, dialysis in the PBS solution, 4 ℃ of storages;
8. the screening of high specific, hypersensitivity cTnI monoclonal antibody
Through said process, finally obtain 16 strain monoclonal antibody strains altogether.Substitute detection with ELISA double antibodies sandwich method, select two strain monoclonal antibody strains.Called after JS09 (promptly being associated with the anti-cTnI monoclonal antibody of vitamin H) and JS05 (promptly being associated with the anti-cTnI monoclonal antibody of horseradish peroxidase), with the anti-cTnI monoclonal antibody of external two strains 2B1.9,2F6.6 (this two strain antibody is at present unique through drugs approved by FDA and most widely used a pair of monoclonal antibody) contrast.People's cardiac muscle, skeletal muscle and the unstriated muscle sample after the fresh homogenate, cTnI, cTnT (TnT) and the cTnC of purifying take a morsel, measure the specific combination of itself and JS09, the strain of JS05 monoclonal antibody by Western Blot method and ELISA double antibodies sandwich method, find that JS09, JS05 monoclonal antibody only combine with the cTnI of people cardiac muscular tissue, and with skeletal muscle, unstriated muscle, cTnC and cTnT no cross reaction.The hypotype of JS09 and JS05 two strain monoclonal antibodies detects through immune competition law, is respectively IgG
1With IgG
2b
(3) the single stage method rapid determination of human body cardiac troponin I
1. detection kit is formed
(1) streptavidin wraps 96 hole elisa plates of quilt in advance, and its set-up procedure is:
A. streptavidin 10 μ g/ml be dissolved in bicarbonate buffer (100mmoL/L, PH9.5), every hole 100 μ l, the bag by 96 hole polyphenyl second elisa plates, 4 ℃, 18 hours.
B. the coating buffer that inclines, with the 1%BSA sealing, 37 ℃, 30min, or 4 ℃, 12 hours.
C. the deblocking liquid that inclines, scavenging solution (containing Tween0.04%) cleans 2 times, preservative film be sealed in 4 ℃ stand-by or-20 ℃ of storages are standby.
(2) the cTnI standard substance are 5 parts, and content is respectively 1,2,4,8,16 μ g/l.
(3) positive and negative quality controlled serum are each 1 part.
(4) the cardiac troponin I monoclonal antibody (JS09-vitamin H) that is associated with vitamin H and each portion of cardiac troponin I monoclonal antibody (JS05-PODs) that is associated with horseradish peroxidase.
(5) antibody and diluted sample damping fluid PBS (phosphoric acid salt 40mmoL/L, PH7.2, NaCL150 mmoL/L) are 1 part.
(6) cleaning buffer solution (Tween0.04%, NaCL 150mmoL/L) is 1 part.
(7) substrate hydrogen peroxide (H
2O
2) with 2, each 1 part in 2 '-Lian oxygen-two-3-ethylbenzene thiazole woods-6-sulfonate (ABTS).
(8) substrate dilution buffer liquid citric acid phosphoric acid salt buffer (100 mmoL/L, PH4.2) 1 part.2. detecting operation process
(1) gets cTnI standard substance (1,2,4,8,16 μ g/l) or testing sample, quality controlled serum 50 μ l/ml, get the cardiac troponin I monoclonal antibody (JS09-vitamin H) that is associated with vitamin H and another cardiac muscle troponin I monoclonal antibody (JS05-PODs) that is associated with respectful peroxidase totally 50 μ l, mixing adds elisa plate and hatches, 37 ℃, 30min, dilution buffer liquid are PBS.
(2) scavenging solution cleans 3 times, adds substrate hydrogen peroxide (H
2O
2) with 2,2 '-Lian oxygen-two-3-ethylbenzene thiazole woods-6-sulfonate (ABTS) 100 μ l, dilution buffer liquid is the citric acid phosphoric acid salt buffer, 37 ℃, 15min.
(3) absorbancy when reading wavelength 405nm is the amount that contained cardiac troponin I in the testing sample is calculated in contrast with standard substance.
3. the correlation parameter in detecting
Reaction reached plateau when (1) the streptavidin bag was by concentration 6 μ g/ml, got streptavidin 10 μ g/ml and be fixed packet by concentration, and the result is stable.Bag was reached maximum by the time in 18 hours at 4 ℃.It is the most stable when bag is cushioned liquid PH9.5.The preservative film sealing is stored January for 4 ℃ does not have statistical discrepancy with-20 ℃ of storage measurement results in June.
(2) cTnI standard substance content is respectively 1,2, during 4,8,16 μ g/L, and straight line correlation parameter r=0.994.
Susceptibility height and result were stable when (3) JS09, JS05 reaction density were respectively 1 μ g/ml and 2 μ g/ml.Reaction is best when antibody and diluted sample damping fluid PBS PH7.2.
(4) substrate hydrogen peroxide (H
2O
2) concentration range is 0.01-0.03%, 2, the background of 2 '-Lian oxygen-two-3-ethylbenzene thiazole woods-when 6-sulfonate (ABTS) concentration is 1.9mmoL/L is low and susceptibility is high.Substrate dilution buffer liquid PH4.0-4.4 is best.Background is low during 37 ℃ of 15min of developing time, and reaction is near plateau.
(5) cTnI standard substance or testing sample, quality controlled serum are hatched 37 ℃ with JS09, JS05 mixing, reach plateau during 30min.
(6) group difference is 8%, and differences between batches are 6%.
(7) 100 routine normal human serum cTnI reference points are 1.5 μ g/L.
Claims (9)
1, the purification process of human body cardiac troponin I is characterized in that comprising following steps:
(1) gets the cardiac muscular tissue of the fresh or quick freezing and cold preserving of human body, degrease and fiber, after step process such as homogenate, centrifugal, precipitation, extracting, high salt dissolving, at chromatography media is CMSephadex-C50, Cellulose-DE52, buffer system is Tris-HCL, pH value is 7.2～7.8, and urea content is to carry out chromatography under the 6-8moL/L condition successively, extracts cardiac troponin C (cTnC);
(2) getting step (1) gained cardiac troponin C, is 0.1～0.3moL/LNaHCO at coupling solution
3, pH value is 7.8～8.5,3～6mmoL/L CaCL
2, level pad is 45～55mmoL/L Tris-HCL, 8～10moL/L urea, 0.8～1.2mmoL/LCaCL
2, 12～18mmoL/L β mercaptoethanol, pH value be under 7.8～8.2 the condition with the coupling mutually of Sepharose 4B affinity column, obtain Sepharose 4B affinity column;
(3) get slightly the carry product of above-mentioned steps (1) behind CM Sephadex-C50 chromatography, add step (2) gained Sepharose 4B affinity column again and carry out affinity chromatography, promptly get the purification product of cardiac troponin I.
2, the method for preparing monoclonal antibody of human body cardiac troponin I according to claim 1 is characterized in that comprising following steps:
(1) gets the prepared cardiac troponin I of aforementioned claim 1, repeatedly inject BALB/c mouse peritoneal immunity mouse, be 2～3 weeks pitch time, to the anti-cardiac troponin I of mice serum antibody titers greater than 1: 2000, get mouse boosting cell and SP2/0 myeloma cell;
(2) with two kinds of cell mixings of above-mentioned steps (1), add the DMEM serum-free medium and handle, add the HAT nutrient solution that contains 15～20% foetal calf serums again, put incubator and cultivate, carry out cytogamy;
(3) get the nutrient solution supernatant of above-mentioned steps (2), detect hybridoma in the nutrient solution, the i.e. specific antibody of cardiac troponin I with the ELISA method;
(4) get the detected hybridoma of above-mentioned steps (3) and adopt limiting dilution assay to cultivate, make the homology cell clone of propagation, i.e. monoclonal cell strain;
(5) the monoclonal cell strain that above-mentioned steps (4) is made contrasts with standard monoclonal antibody 2B1.9,2F6.6, adopts Western blot and ELISA method to measure, and detects anti-cardiac troponin I and catches monoclonal antibody IgG
1(being associated with the monoclonal antibody of horseradish peroxidase) and anti-cardiac troponin I mark biotin antibody IgG
2b(being associated with the monoclonal antibody of vitamin H), above-mentioned two monoclonal antibodies are the monoclonal antibody of the cardiac troponin I with high specific, hypersensitivity of present method gained.
3, the single stage method rapid assay methods of human body cardiac troponin I is characterized in that comprising following steps:
(1) with 50～150mmoL/L, pH value is that 9.4～9.6 bicarbonate buffer, streptavidin bag are by 96 hole polystyrene elisa plates;
(2) get testing sample and standard substance, with after the monoclonal antibody of the anti-cardiac muscle troponin I that is associated with vitamin H and the monoclonal antibody that is associated with the anti-cardiac troponin I of horseradish peroxidase contact, add above-mentioned elisa plate bag respectively respectively by in the hole;
(3) elisa plate of step (2) is hatched;
(4) get elisa plate after hatching, add substrate hydrogen peroxide and 2 at its bag respectively in by the hole, 2 '-Lian oxygen-two-3-ethylbenzene thiazole woods-6-sulfonate carries out enzymatic and develops the color;
Absorbancy when (5) reading above-mentioned steps (4) enzymatic colour developing back wavelength 405nm contrasts testing sample and standard substance, calculates the content of the cardiac troponin I in the testing sample.
4, according to the arbitrary described method of claim 1～3, cardiac troponin C, the I that it is characterized in that purifying derives from the people cardiac muscular tissue of-75 ℃ of storages behind fresh or the liquid nitrogen flash freezer.
5,, it is characterized in that anti-cTnI monoclonal antibody derives from the fused cell of BALB/c mouse and SP2/0 murine myeloma cell according to the arbitrary described method of claim 1～3.
6, according to claim 2 or 3 described methods, it is characterized in that used anti-cTnI monoclonal antibody is the anti-cTnI monoclonal antibody that is associated with the anti-cTnI monoclonal antibody of vitamin H and is associated with horseradish peroxidase, this two anti-cTnI monoclonal antibody is respectively IgG
1With IgG
2bHypotype.
7,, it is characterized in that the anti-cTnI monoclonal antibody that is associated with the anti-cTnI monoclonal antibody of vitamin H and is associated with horseradish peroxidase is to derive from mouse according to claim 2 or 3 described methods.
8, method according to claim 3, it is characterized in that testing sample and standard substance are between PH6.5-7.5, the phosphate buffered saline buffer of preferred PH7.2 contacts with anti-cTnI monoclonal antibody that is associated with vitamin H and the anti-cTnI monoclonal antibody that is associated with horseradish peroxidase.
9, method according to claim 3, employed washings pH value is between 6.8-8 before to it is characterized in that colour developing, and temperature preferably is lower than 10 ℃ at 2-37 ℃.
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| CN 03154208 Division CN1247615C (en) | 2000-03-21 | 2000-03-21 | Purification of human myocardium troponin I and preparation method of its monoclonal anti-body |
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| CN1165548C CN1165548C (en) | 2004-09-08 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101942416A (en) * | 2010-07-23 | 2011-01-12 | 中国医学科学院放射医学研究所 | Anti-human cardiac troponin I specific monoclonal antibody and preparation method thereof |
| CN101105497B (en) * | 2006-07-13 | 2011-10-19 | 上海凯创生物技术有限公司 | Cardiac muscle troponin I detection reagent kit and its preparation method |
| CN104894652A (en) * | 2015-06-25 | 2015-09-09 | 黄薇 | Construction and application of humanized single-chain antibody libraries of cTnI (cardiac troponin I) |
| CN110272502A (en) * | 2019-07-12 | 2019-09-24 | 深圳市亚辉龙生物科技股份有限公司 | The hybridoma and preparation method, monoclonal antibody and application of immunogene, the anti-cardiac muscle troponin I monoclonal antibody of secretion |
| CN114206920A (en) * | 2019-08-09 | 2022-03-18 | 豪夫迈·罗氏有限公司 | Novel anti-troponin T antibodies |
| CN114206920B (en) * | 2019-08-09 | 2024-06-07 | 豪夫迈·罗氏有限公司 | Novel anti-troponin T antibodies |
-
2000
- 2000-03-21 CN CNB001121561A patent/CN1165548C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101105497B (en) * | 2006-07-13 | 2011-10-19 | 上海凯创生物技术有限公司 | Cardiac muscle troponin I detection reagent kit and its preparation method |
| CN101942416A (en) * | 2010-07-23 | 2011-01-12 | 中国医学科学院放射医学研究所 | Anti-human cardiac troponin I specific monoclonal antibody and preparation method thereof |
| CN104894652A (en) * | 2015-06-25 | 2015-09-09 | 黄薇 | Construction and application of humanized single-chain antibody libraries of cTnI (cardiac troponin I) |
| CN110272502A (en) * | 2019-07-12 | 2019-09-24 | 深圳市亚辉龙生物科技股份有限公司 | The hybridoma and preparation method, monoclonal antibody and application of immunogene, the anti-cardiac muscle troponin I monoclonal antibody of secretion |
| CN114206920A (en) * | 2019-08-09 | 2022-03-18 | 豪夫迈·罗氏有限公司 | Novel anti-troponin T antibodies |
| CN114206920B (en) * | 2019-08-09 | 2024-06-07 | 豪夫迈·罗氏有限公司 | Novel anti-troponin T antibodies |
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| Publication number | Publication date |
|---|---|
| CN1165548C (en) | 2004-09-08 |
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